SPID#: 9 Recombinant vaccinia viruses expressing full length or truncated gag or env genes of SIVmac239 are used to investigate the requirements for assembly of SIV proteins. Assembly of virus-like particles (VLPs) was found to be 3 to 5 fold higher with full length env than with the truncated forms, or than VLPs containing only Gag proteins, in primary monkey cells or various human cell lines. When cells expressing Env proteins in the absence of Gag were examined by immunoelectron microscopy, clusters of Env protein and membrane vesicles containing Env proteins were observed at cell surfaces. A low level of vesicles was released from cells expressing full length Env, but about a 10-fold higher level was released in cells expressing a truncated form of Env [Env733(t)] in which the cytoplasmic domain is only 17 amino acids in length. Another truncated protein, Env718(t), with a short cytoplasmic tail of 3 aa was also incorporated into VLPs at a 10-fold higher level than the full length Env protein, and was more efficiently released in vesicles. The mature SU and TM proteins were predominantly incorporated into VLPs with full length Env, but both cleaved and uncleaved precursor proteins were present in VLPs with truncated Env as well as in Env and Env(t) vesicles. A more prominent layer of spikes was seen by electron microscopy in VLPs with truncated Env than in VLPs containing full length Env. These results indicate that truncated Env proteins have the ability to self-associate on the cell surface and are assembled into a more closely packed array than full length Env, which could explain the preferential incorporation of Env proteins with short cytoplasmic tails into virions.